Use of antitumoral compound in cancer therapy

ABSTRACT

Improved dosing schedules for ecteinascidin 743 are given for treatment of cancer.

FIELD OF THE INVENTION

The present invention is directed to the use of ecteinascidin 743 andproducts containing this compound for cancer therapy, in particular toimprovements in the use of ecteinascidin 743 in the treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer comprises a group of malignant neoplasms that can be divided intotwo categories, carcinoma, comprising a majority of the cases observedin the clinics, and other less frequent cancers, which include leukemia,lymphoma, central nervous system tumours and sarcoma. Carcinomas havetheir origin in epithelial tissues while sarcomas develop fromconnective tissues and those structures that had their origin inmesoderm tissues. Sarcomas can affect, for instance, muscle or bone andoccur in the bones, bladder, kidneys, liver, lung, parotid, spleen, etc.

Cancer is invasive and tends to metastasise to new sites. It spreadsdirectly into surrounding tissues and also may be disseminated throughthe lymphatic and circulatory systems.

Many treatments are available for cancer, including surgery andradiation for localised disease, and drugs. However, the efficacy ofavailable treatments on many cancer types is limited, and new, improvedforms of treatment showing clinical benefit are needed. This isespecially true for those patients presenting with advanced and/ormetastatic disease. It is also true for patients relapsing withprogressive disease after having been previously treated withestablished therapies for which further treatment with the same therapyis mostly ineffective due to acquisition of resistance or to limitationsin administration of the therapies due to associated toxicities.

Chemotherapy plays a significant part in cancer treatment, as it isrequired for treatment of advanced cancers with distant metastasis andoften helpful for tumor reduction before surgery, and many anti-cancerdrugs have been developed based on various modes of action.

The ecteinascidins (herein abbreviated Et or Et's) are exceedinglypotent antitumor agents isolated from the marine tunicate Ecteinascidiaturbinata. Several ecteinascidins have been reported previously in thepatent and scientific literature. See, for example U.S. Pat. No.5,256,663, which describes pharmaceutical compositions comprising matterextracted from the tropical marine invertebrate, Ecteinascidiaturbinata, and designated therein as ecteinascidins, and the use of suchcompositions as antibacterial, anti-viral, and/or antitumor agents inmammals; U.S. Pat. No. 5,089,273, which describes novel compositions ofmatter extracted from the tropical marine invertebrate, Ecteinascidiaturbinata, and designated therein as ecteinascidins 729, 743, 745, 759A,759B and 770. These compounds are useful as antibacterial and/oranti-tumor agents in mammals; U.S. Pat. No. 5,478,932, which describesecteinascidins isolated from the Caribbean tunicate Ecteinascidiaturbinata, which provide in vivo protection against P388 lymphoma, B16melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and the LX-1human lung and MX-1 human mammary carcinoma xenografts.

One of them, ecteinascidin-743 (ET-743), is a noveltetrahydroisoquinoline alkaloid isolated from the marine ascidianEcteinascidia turbinata that has considerable antitumor activity inmurine and human tumors in vitro.

In a study of human cancer cell lines, ET-743 exhibited extremely potentactivity against several soft tissue sarcoma cell lines with IC₅₀s wellbelow 1 pM. See for example Li W, Jhanwar S, Elisseyeff Y, Bertino J R.“Potent antitumor activity of ET-743 against human soft tissue sarcomacell lines”, Clin Cancer Res 1999; 5: 305 and Izbicka E, Lawrence R,Raymond E, et al.: “In vitro antitumor activity of the novel marineagent, Ecteinascidin-743 against human tumors explanted from patients”,Ann. Oncol. 1998; 9: 981-7.

Potent antitumor activity has been demonstrated in a broad range of invivo tumor models, including human tumor xenografts in nude mice. Thisis illustrated in Valoti G, Nicoletti M I, Faircloth G, et al.:“Antitumor effect of ecteinascidin-743 (ET-743) on human ovariancarcinoma xenografts”, Proc. Am. Assoc. Cancer Res. 1997; 38: 1477;Faircloth G, Hendriks H R, Giavazzi R, et al.: “In vivo antitumoractivity of Ecteinascidin 743 (ET 743), a novel marine derived cytotoxicagainst human xenografts tumor models”, Ann Oncol 1996; 7: 125; HendriksH R, Fiebig H H, Giavazzi R, et al.: “High antitumour activity of ET743against human tumour xenografts from melanoma, non-small-cell lung andovarian cancer” Ann. Oncol. 1999; 10: 1233-40.

Et-743 has a novel complex mechanism of action at the level of genetranscription. ET-743 binds to guanine-cytosine rich sequences in theminor groove of DNA and alkylates guanine residues at the N2 position,see Pommier Y, Kohlhagen G, Ballly C, et al.: “DNA sequence- andstructure-selective alkylation of guanine N2 in the DNA minor groove byEcteinascidin 743, a potent antitumor compound from the Caribbeantunicate Ecteinascidia turbinata”, Biochemistry 1996; 35: 13303-9. Cellcycle studies have demonstrated that ET-743 decreases the rate ofprogression of tumor cells through S-phase and causes prolongedp53-independent blockade in G₂/M, giving rise to a strong apoptoticresponse, Erba W. Bergamaschi D, Ronzoni S, et al.: “Mode of action ofEcteinascidin 743, a natural marine compound with antitumor activity”Ann. Oncol. 1998; 9: 535. Cells in G₁ are more sensitive to thecytotoxic effects of ET-743 than cells in S-phase or G2/M. These effectsappear to be mediated by multiple mechanisms. ET-743 strongly inhibitsthe activation of the transcription of certain genes, including p21,c-fos, c-jun and mdr1, without affecting their basal transcriptionlevels. Further background concerning this point is to be found inMantovani R, La Valle E, Bonfanti M, et al.: “Effect of ET-743 on theinteraction between transcription factors and DNA”, Ann. Oncol. 1998; 9:534; Minuzzo M, Marchini S, Broggini M, et al.: “Interference oftranscriptional activation by the antineoplastic drugecteinascidin-743”, Proc. Natl. Acad. Sci. USA 2000; 97: 6780-4; Jin S,Gorfajn B, Faircloth G, Scotto K W.: “Ecteinascidin 743, atranscription-targeted chemotherapeutic that inhibits MDR1 activation”,Proc. Natl. Acad. Sci. USA 2000; 97: 6775-9; Synold T W, Dussault I,Forman B M.: “The orphan nuclear receptor SXR coordinately regulatesdrug metabolism and efflux”, Nat. Med. 2001; 7: 584-90.

Toxicological evaluations of ET-743 as a single or a fractionated doseby the intravenous route in mice, rats, and dogs have consistently shownthe potential of ET-743 to induce reversible hematological and hepatictoxicity. Liver toxicity was evident from transient increase in serumlevels of liver enzymnes, bilirubin and bile acids, and fromhistopathological changes in the liver. Further toxicity includedlesions at the site of injection, spleen and thymus lesions, bile ducthyperplasia, portal fibrosis, gall bladder lesions characterized bycholecystitis with oedema and a lymphocytic infiltrate, pancreaticacinar cell atrophy and apoptosis, and decreased testicular and ovarianweights. Studies in dogs showed vomiting and diarrhoea following theadministration of ET-743. A study in cynomolgus monkeys confirmed thepotential of single doses of ET-743 to induce hepatic and hematologicaltoxicity, emesis and diarrhoea. However, fractionated dosing inducedonly minor toxicity in monkeys. See Jimeno J, Faircloth G, Cameron L, etal.: “Progress in the acquisition of new marine.derived anticancercompounds: development of ecteinascidin-743 (ET-743)”, Drugs Future1996; 21: 1155-65.

An in vitro bone marrow assay using human, murine and canine progenitorcells, showed equal sensitivity of erythropoid and myeloid cells toET-743. Prolonged or repeated exposure to the drug proved more toxic tohematopoietic progenitors than a single 1-hour exposure, see for exampleGhielmini M, Colli E, Erba E, et al.: “In vitro schedule-dependency ofmyelotoxicity and cytotoxicity of Ecteinascidin 743 (ET-743)”, Ann.Oncol. 1998; 9: 989-93. The therapeutic index of ET-743 was morefavourable with prolonged exposure.

A clinical development program of ET-743 in cancer patients was startedwith phase I studies investigating 1-hour, 3-hour, 24-hour and 72-hourintravenous infusion schedules and a 1 hour daily×5 (d×5) schedule.These studies are reported in Taamma A, Misset J L, Riofro M, et al.:“Phase I and pharmacokinetic study of ecteinascidin-743, a new marinecompound, administered as a 24-hour continuous infusion in patients withsolid tumors”, J. Clin. Oncol. 2001; 19: 1256-65; Van Kesteren C,Cvitkovic E, Taamma A, et al.: “Pharmacokinetics and pharamacodynamicsof the novel marine derived anticancer agent ecteinascidin 743 in aphase I dose-finding study”, Clin. Cancer Res. 2000; 6: 4725-32; Ryan DP, Supko J G, Eder J P, et al.: “Phase I and pharmacokinetic study ofecteinascidin 743 administered as a 72-hour continuous intravenousinfusion in patients with solid malignancies” Clin. Cancer Res. 2001; 7:231-42; Villalona-Calero M A, Eckhardt S G, Weiss G, et al.: “A phase Iand pharmacokinetic study of ecteinascidin-743 on a daily×5 schedule inpatients with solid malignancies”, Clin. Cancer Res. 2002; 8: 75-85.Further detail on the use of Et-743 for the treatment of the human bodyfor cancer is given in WO 0069441.

Summary results of each phase I study are detailed below:

24hr infusion every 3 weeks: A total of 52 patients were treated at ninedifferent dose levels and received a total of 158 cycles. The MTD(maximum tolerated dose) and the RD (recommended dose) were determinedat 1800 μg/m² and 1500 μg/m². The DLT (dose limiting toxicity) washematotoxicity (i.e., neutropenia and thrombocytopenia). At the RD,transient and reversible increases in transaminases were observed inmost patients. Grade 3 or 4 transaminases occurred in 68% of patientsand 38% of cycles. Grade 2 or greater hyperbilirubinemia was reported in30% of the treatment courses at the RD. Other toxicities includednausea/vomiting and asthenia.

72 hr infusion every 3 weeks: In this study, 21 adult patients wereevaluated at four dose levels of ET-743. Non-hematological dose-limitingtoxicity with 1200 μg/M² (MTD) was reversible grade 4 transaminaseselevation (in 2 out of 9 patients), which was considered a DLT in thisstudy. A third patient at this dose level experienced grade 4rhabdomyolysis, grade 4 febrile neutropenia, and grade 4thrombocytopenia. At the RD (1050 μg/M²) there was not any grade 4hypertransaminasemia, and grade 3 was reported in 50% of patients.Increases in bilirubin and alkaline phosphatase occurred less often andwere below grade 2. Nausea/vomiting and fatigue were also reported .Prolonged infusion of ET-743 over 72 hours did not allow theadministration of a higher total dose per cycle than over 24 hours.

1 hr infusion days 1-5 every 3 weeks: 42 patients were treated at 10dose levels ranging from 6 to 380 μg/m²/day. At the 380 μg/m²/day doselevel (MTD), 3 patients developed DLT gong-lasting neutropenia) and oneof them died because of toxicity. At the 325 μg/m²/day dose level (RD),59% of cycles were delayed, most of them because of treatment relatedtoxicity. Furthermore, the median day of recovery to grade 1 (at whichtreatment could be recommended) for neutropenia was 28 days. Thus, insome patients it may be necessary to administer the RD of 325 μg/m²/dayET-743 on a 4 week schedule. There were not any grade 4 transaminasestoxicities even at the MTD. Reversible grade 3 transaminases occurred in14% of cycles and 31% of patients at the recommended dose. An event ofgrade 3 hyperbilirubinemia occurred at the RD.

1 and 3 hr infusion every 3 weeks: In the first part of the study 40patients were treated with ET-743 given as a 1-hr intravenous infusionevery 3 weeks. A maximum tolerated dose (MTD) of 1100 μg/m² was defined.The dose limiting toxicities (DLT) were: grade 4 fatigue, grade 4neutropenia lasting more than 5 days and grade 4 thrombocytopenia. Atthe MTD level, emesis and grade 3-4 transaminases elevation, which insome cycles did not recover by day 21, were observed too. It wasrealized that the MTD and recommended dose (RD) in this study were muchlower than in a concurrent study with ET-743 given as a 24-hr continuousinfusion. Since the latter schedule requires in-patient treatment, itwas felt that a shorter infusion time was preferable if the same doselevel could be reached. Therefore, the protocol was amended to assessthe feasibility of ET-743 given as a 3-hr infusion. Prolonging theinfusion from 1 to 3 hours resulted in a higher MTD and RD for Phase IIstudies (1800 and 1650 μg/M²) with a similar toxicity profile. 32patients were treated in the second part of the study. DLTs includedagain G4 thrombocytopenia and G3 fatigue. Other toxicities includedelevation of transaminases and increases of bilirubin G2 or greater in9% of treatment courses at the RD. Decreases of leukocytes andneutrophil counts, asthenia, nausea/vomiting and phlebitis were alsoobserved. Liver toxicity was evident in many patients from markedincreases in serum ALT and AST and signs of cholestasis as evidenced byincreases in alkaline phosphatase and bilirubin, although these occurredless frequently and were of lower grade than the increases intransaminases.

Two schedules (24 hour every 3 weeks and 3 hour every 3 weeks) reachedthe phase II programme. The phase II programme confirmed the activityagainst soft tissue sarcoma and ovarian cancer. However, the recommendedstarting dose for the 3 hour schedule had to be reduced because ofserious toxicity. The recommended dose for the 24 hour schedule was 1500μg /m² and the recommended dose for the 3 hour schedule is at present1300 μg/m².

Serious increases of transaminases (grade 3-4) were more frequent withthe 3 hour schedule than with the 24 hour schedule. Increasedtransaminases were seen in 83.4% of patients and 58.3% of cycles. Thistoxicity has improved with the amendment that reduces the starting doseto 1300 (grade 3 and 4 ALT in 38% and 8.5% of patients), although itwould be desirable to reduce it further. Cholestasis is less severe andless frequent observing grade 1, 2 and 3 alkaline phosphatase in 50.4%,6.5% and 1.7% (leading to an overall 58.6% that compares similarly with57.7% over 24 hours) of patients. Grades 1-3 of bilirubin in 45% ofpatients was observed (versus 23.8% of patients receiving the drug over24 hours). An infrequent but important toxicity was the renal toxicity,represented by creatinine abnormalities, that is also higher than in the24 hour schedule.

The 3 hour every three weeks schedule has the signifcant advantage ofbeing more confortable for the patient because it reduces the time beingspent at the hospital for infusion and monitoring, in particularavoiding overnight stay. However, the schedule exhibited a greatertoxicity as mentioned above and as illustrate by the following tables:Hematological Toxicity. Worst grade per patient. 24 hour infusionNCI-CTC Grade-Number of patients (%) N 0-1 2 3 4 Neutrophils 319 103(32.3)  54 (16.9) 92 (28.8)  70 (21.9) Platelets 319 260 (81.5) 17 (5.3)35 (11.0)  7 (2.2) Hemoglobin 320 165 (51.6) 111 (34.7) 33 (10.3) 11(3.4)

Liver Toxicity. Worst Grade Per Patient. 24 hour Infusion NCI-CTCGrade-Number of patients (%) N 1 2 3 4 Bilirubin 320 49 (15.3) 23 (6.9)  4 (1.3) Alk Phosph 319 149 (46.7)  29 (9.1)   6 (1.9) Gamma GT 109 25(22.9) 29 (26.6)  32 (29.4)  2 (1.8) SGOT/AST 319 74 (23.2) 88 (27.6)120 (37.6) 16 (5.0) SGPT/ALT 320 62 (19.4) 83 (25.9) 127 (39.7) 30 (9.4)

Hematological Toxicity. Worst grade per patient. 3 hour infusion NCI-CTCGrade-Number of patients (%) N 0-1 2 3 4 Neutrophils 243  59 (24.5) 42(17.4) 57 (23.6) 85 (35.2) Platelets 241 170 (69.1) 26 (10.6) 31 (12.6)14 (5.6)  Hemoglobin 239 132 (55)   80 (33.4) 23 (9.6)  4 (1.6)

Liver toxicity. Worst grade per patient. 3 hour infusion NCI-CTCGrade-Number of patients (%) N 1 2 3 4 Bilirubin 228  59 (25.8) 38(16.6)  6 (2.6) 0      Alk Phosph 228 115 (50.4) 15 (6.5)   4 (1.7) 2(0.8) SGOT/AST 232 17 (7.2) 29 (12.3) 117 (49.7) 58 (24.6) SGPT/ALT 23313 (5.5) 19 (8.1)  107 (45.7) 87 (37.1)

Creatinine abnormalities. 3 hour infusion NCI-CTC Grade N 1 2 3 4 Perpatient 218 76 (23.9) 19 (6.0) 3 (0.9) 2 (0.6) (24 h) Per patient 218 44(20.1) 10 (4.6) 5 (2.3) 0      (3 h)

It is an object of the present invention to provide a cancer therapyusing Et-743 which allows for short infusion times while minimizingtoxicities induced by the administration of ET-743, and withoutsacrificing the desired antineoplastic effects.

SUMMARY OF THE INVENTION

We have now found, unexpectedly, that a different type of schedule anddosage allows for an effective cancer therapy with ET-743. Surprisingly,our results show that it is possible to administer ET-743 with reducedinfusion times while avoiding toxicities and mantaining the desiredantineoplastic effects. It is significant that the most frequenttoxicities have been reduced more than 3 times, transaminases toxicityis reduced up to 8 times less than the 3 hour every three weeks scheduleand serious renal toxicity is avoided.

The present invention provides a method of treating cancer in humans,comprising intravenously infusing a composition comprising ET-743 into ahuman having cancer at continuous dosage over a period up to 4 hours,wherein the step of infusing is repeated weekly on a cyclic basis.

The infusing step is typically repeated on a cyclic basis. The cyclicbasis comprises two phases, the phase of weekly infusing and a phase ofnot infusing, referred to a rest phase. In the rest phase the patientsare allowed to recover. Usually the cycle is worked out in weeks, andthus the cycle comprises one or more weeks of an infusion phase, and oneor more weeks of a rest phase. Preferably the rest period is not longerthan the infusion phase. Thus, preferably the rest phase is the samenumber of weeks as the infusion phase, or a lesser number of weeks.Particularly preferred is for the infusion phase to be a greater numberof weeks than the rest phase, though a cycle of one week infusion andone week rest is envisaged. Preferably the resting phase is one weekwithin each cycle. The preferred duration of each cycle is of 2 to 4weeks; multiple cycles can be given as needed. A cycle of 4 weeks ismost preferred.

In a particular embodiment, the infusion time is between 1 and 3 hours,preferably between 2 and 3 hours. Especially preferred is a time ofabout 3 hours.

In another embodiment of the invention, the dosage of Et-743 is below650 μg/m²/weekly, preferably between 300 and 600 μg/m²/weekly, morepreferably between 400 and 600 μg/m²/weekly. Suitably the dosage isbetween 525 and 600 μg/m²/weekly, especially preferred is a dosage ofabout 580 μg/m²/weekly.

The above schedules and dosages allow for an effective cancer therapy inhumans, while avoiding toxicities. This means that with these dosagesand schedules the therapuetic index is improved. We have found thatET-743 is effective in the treatment of several cancer types, includingadvanced or metastatic. Preferably, ET-743 is used according to theabove schedules and dosages for the treatment of sarcoma, osteosarcoma,ovarian cancer, breast cancer, melanoma, colorectal cancer,mesothelioma, renal cancer, endometrial cancer and lung cancer.

The present invention also provides a pharmaceutical compositioncontaining a recommended dose of ET-743 for weekly administration and apharmaceutically acceptable carrier.

In a further aspect of the present invention, a medical kit foradministering ET-743 is provided, comprising printed instructions foradministering ET-743 according to the dosing schedules set forth above,and a supply of ET-743 in dosage units for at least one cycle, whereineach dosage unit contains the appropriate amount of ET-743 for thetreatments as defined above and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION

ET-743 is a natural compound represented by the following formula:

ET-743 is supplied and stored as a sterile lyophilized product,consisting of ET 743 and excipient in a formulation adequate fortherapeutic use, in particular a formulation containing mannitol and aphosphate salt buffered to an adequate pH.

A preferred formulation, which shows improved stability at higherstorage temperature, is one which after dilution contains per ml. 0.05mgof ET743, 50 mg of mannitol and 6.8 mg of potassium dihydrogen phosphateto adjust to a pH between 4.00 and 6.00, with 4.80 being the preferredpH. The product is lyophilized and stored in the cold, between +4 C and−20 C and protected from light until use.

Preparation of the reconstituted solution is performed under asepticconditions by adding distilled water in the amount of 5 ml for every0.25 mg of ET-743 and shaking for a short time to dissolve the solids.

Preparation of the infusion solution is also performed under asepticconditions by withdrawing the reconstituted solution volumecorresponding to dosage calculated for each patient, and slowlyinjecting the required reconstituted solution volume into an infusionbag or bottle containing between 100 and 1000 ml of 0.9% sodium chloridesolution, after which the whole is homogenised by slow manual shaking.The ET-743 infusion solution should be administered intravenously, assoon as possible, within 48 hours after preparation. PVC andpolyethylene infusion systems, as well as clear glass are preferredcontainer and conduit materials.

The administration is performed in cycles, in the application method ofthe invention, an intravenous infusion of ET743 is given to the patientsevery week, allowing for a resting phase in each cycle in which thepatients recover. Preferably the resting phase is one week within eachcycle. The preferred duration of each cycle is of 2 to 4 weeks; multiplecycles can be given as needed. Dose delays and/or dose reductions andschedule adjustments are performed as needed depending on individualpatient tolerance of treatments, in particular does reductions arerecommended for patients with higher than normal serum levels of livertransaminases or alkaline phosphatase, or bilirubin.

Depending on the type of tumour and the developmental stage of thedisease, the treaments of the invention are useful in preventing therisk of developing tumours, in promoting tumour regression, in stoppingtumour growth and/or in preventing metastasis.

Although guidance for the dosage is given above, the correct dosage ofthe compound will vary according to the particular formulation, the modeof application, and the particular situs, host and tumour being treated.Other factors like age, body weight, sex, diet, time of administration,rate of excretion, condition of the host, drug combinations, reactionsensitivities and severity of the disease shall be taken into account.Administration can be carried out continuously or periodically withinthe maximum tolerated dose.

The Recommended Dose (RD) is the highest dose which can be safelyadministered to a patient producing tolerable, manageable and reversibletoxicity according to the Common Toxicity Criteria (CTC) established forexample by the National Cancer Institute, (USA) typically with no morethan 2 out of 6 patients presenting any dose limiting toxicities (DLT).Guidelines for cancer therapy frequently call for administration ofchemotherapeutic agents at the highest safe dose at which toxicity ismanageable in order to achieve maximum efficacy (DeVita, V. T. Jr.,Hellman, S. and Rosenberg, S. A., Cancer: Principles and Practice ofOncology, 3rd ed., 1989, Lipincott, Philadelphia). For ET-743, therecommended doses are as defined above and set forth in the examples.

The compound ET743 and compositions of this invention may be used withother drugs to provide a combination therapy. The other drugs may formpart of the same composition, or be provided as a separate compositionfor administration at the same time or a different time. The identity ofthe other drug is not particularly limited, and suitable candidatesinclude:

-   a) drugs with antimitotic effects, especially those which target    cytoskeletal elements, including microtubule modulators such as    taxane drugs (such as taxol, paclitaxel, taxotere, docetaxel),    podophylotoxins or vinca alkaloids (vincristine, vinblastine);-   b) antimetabolite drugs (such as 5-fluorouracil, cytarabine,    gemcitabine, purine analogues such as pentostatin, methotrexate);-   c) alkylating agents or nitrogen mustards (such as nitrosoureas,    cyclophosphamide or ifosphamide);-   d) drugs which target DNA such as the antracycline drugs adriamycin,    doxorubicin, pharmorubicin or epirubicin;-   e) drugs which target topoisomerases such as etoposide;-   f) hormones and hormone agonists or antagonists such as estrogens,    antiestrogens (tamoxifen and related compounds) and androgens,    flutamide, leuprorelin, goserelin, cyprotrone or octreotide;-   g) drugs which target signal transduction in tumour cells including    antibody derivatives such as herceptin;-   h) alkylating drugs such as platinum drugs (cis-platin,    carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;-   i) drugs potentially affecting metastasis of tumours such as matrix    metalloproteinase inhibitors;-   j) gene therapy and antisense agents;-   k) antibody therapeutics;-   l) other bioactive compounds of marine origin, notably the didemnins    such as aplidine;-   m) steroid analogues, in particular dexamethasone;-   n) anti-inflammatory drugs, including nonsteroidal agents (such as    acetaminophen or ibuprofen) or steroids and their derivatives in    particular dexamethasone; and-   o) anti-emetic drugs, including 5HT-3 inhibitors (such as    gramisetron or ondasetron), and steroids and their derivatives in    particular dexamethasone.

Particularly preferred for use in combination therapy are dexamethasone,doxorubicin, cisplatin, paclitaxel and dexamethasone. Further guidanceon combination therapy is given in WO 0236135, incorporated herein byreference in its entirety.

EXAMPLES

A phase I clinical trial was carried out with the following protocol:

PROTOCOL:

Dose schedule: ET-743 will be administered every week as a 3hours ivinfusion for 3 consecutive weeks every 4 weeks.

Starting dose and dose escalation: The starting dose will be 300 μg/m²weekly of ET-743 given as a intravenous infusion over 3 hours for 3consecutive weeks every 4 weeks. Patients will be sequentially enrolledinto the following dose cohorts beginning at dose Level 1. A minimumcohort size of 3 patients will be treated at each of the dose levels.ET-743 Dose Escalation Scheme Dose Level −1 200 μg/m² weekly Dose Level1 300 μg/m² weekly Dose Level 2 400 μg/m² weekly Dose Level 3 525 μg/m²weekly Dose Level 4 650 μg/m² weekly Dose Level 5 775 μg/m² weekly DoseLevel 6 900 μg/m² weeklyAccrual at the next higher dose level: At least 1 patient at each doselevel must have completed 1 cycle of therapy and two patients must havecompleted treatment on day 15 before a new patient can be treated at thenext highest dose.Conditions for retreatment: Patients are eligible for retreatment withintolerable toxicity, the patient desires further treatment, and fulfillthe eligibility criteria.Dose Limiting Toxicities (DLTs)

Dose limiting toxicities (DLT) will be defined as follows:

-   -   ANC <500/μL for longer than 5 days.    -   ANC <500/μL accompanied by fever (at least 100.5° F.).    -   Platelets <25,000/μL.    -   Any grade 3-4 nonhematological toxicity except nausea/vomiting        (provided the patient is receiving an optimal antiemetic regimen        consisting of dexamethasone and a serotonin antagonist on an        optimal dose-schedule for prophylaxis and management), alopecia.        Grade 3 or 4 elevation in trasaminases that result in either        omission of 2 scheduled treatments within a cycle or delay in        the initiation of a subsequent course exceeding 2 weeks.    -   Missing at least 2 scheduled treatments during a single course        due to drug-induced toxicity (missed doses will not be made up).    -   Delay in the initiation of a subsequent course of treatment        exceeding 2 weeks.        Determination of the Maximum Tolerated Dose (MTD)

Cohort of 3 patients will be treated at each dose level. If no DLT isseen during the first cycle in the cohort of patients at any given doselevel, new patients may be treated at the next higher level.

If any patient encounters drug-induced DLT during either cycle 1 or 2, amaximum of 6 patients may be treated at that level. If DLT is notobserved in the additional patients, new patients may be treated at thenext higher dose level.

If at least 2 patients experience DLT at any given dose level, this doselevel will be considered the Maximum Tolerated Dose (MTD). However, itis possible that additional patients may experience DLT due to thetiming of patient enrollment into that dose level.

Recommended Dose for phase II studies (RD)

Once an MTD level is established, subsequent patients should be treatedat the next lower dose level. Intermediate doses may be used in someinstances and flexibility is an integral part of the protocol. If two ormore patients experience DLT at the lower dose level, then the MTD hasagain been established and additional patients will be treated at thenext lower dose (unless sufficient numbers of patients have already beentreated at that dose level).

The RD is defined as the highest dose level at which less than 2 of 6patients experience DLT during cycles 1 or 2. At the RD, sufficientnumbers of patients will be accrued so at least 6 patients receive atleast 2 cycles of therapy, and at least 4 patients receive at least 4courses of treatment.

Results:

This trial was launched in May 00 and last patient was included in March02. 31 patients were treated.

Tumours included: Sarcomas (19), UOT (1), Lung (1), Ovary (4), Breast(2), Uterus (1), Melanoma (2), Colorectal (1) Dose level Patients 300 3400 3 525 4 650 6 580 15

2 DLT defined the MTD in this trial: Long lasting grade 3 neutropenia,and g3 Bilirubin toxicity. Both DLTs were found at the 4th level. So,the MTD in this trial was 650 mcg/sm weekly×3/4 weeks. The recommendeddose is 580 mcg/sm×3 every 4 weeks.

Toxicities

After evaluating 29 patients, grade 3-4 neutropenia was 10.3% perpatient and G3 transaminases 10%.

The following tables display the main features of the toxicity seen withthis schedule. Hematologic toxicity per patient. 3 hour weekly ×3/4weeks infusion Number patients G2(%) G3(%) G4(%) Hemoglobin 29 9(31)1(3.4) 0 Platelets 29 1(3.4) 0 0 Neutrophils 29 1(3.4) 2(6.9) 1(3.4)

Nonhematologic toxicity per patient. 3 hour weekly ×3/4 weeks infusionNumber patients G2(%) G3(%) G4(%) Creatinine 29 1(3.4) 0 0 Creatine 291(3.4) 0 1(3.4) Kinase Bilirubin 29 0 1(3.4) 0 ALT 29 9(31) 3(10.3) 0Alk Phos 29 1(3.4) 2(6.9) 0

According to the previously depicted toxicity, this schedule shows anexcellent profile of toxicity, improving the previous one (obtained withthe 24 and 3 hours schedule every three weeks). It can be seen on thetable that neutropenia, thrombopenia, transaminases increases andcreatinine (the most frequent adverse events) have now a much lowerfrequency.

Bilirubin and creatinin-kinase seem to be higher than with the previousones. 3.4% in the weekly trial means that 1 patient had this toxicityand could be unrepresentative. More patients need to be treated toconfirm if this will be the true incidence. Grade 3-4 24 h 3 h WeeklyHemoglobin 13.7 11.3 3.4 Platelets 13.2 18.7 0 Neutrophils 50.7 58.310.3 Creatinine 0.7 2.3 0 Creatinine 2.2 1.5 3.4 Kinase Bilirubin 1.32.6 3.4 ALT 49.1 82.8 10.3

Activity

Some hints of activity were seen:

2 minor responses in patients with sarcoma and long lastingstabilizations in sarcoma (2) and ovarian cancer (2).

Conclusions

From the clinical point of view the weekly schedule is really attractivein terms of toxicity. The most frequent toxicities have been reducedmore than 3 times. And for instance transaminases toxicity is 8 timesless than the 3 hour schedule.

Serious renal toxicity (G3-4) has not been seen with this schedule.

This schedule is very well tolerated and toxicities have been minimizedin an profitable way. Though phase I trial are not designed to evaluateefficacy some hints of activity were seen.

1. A method of treating cancer in humans, comprising intravenouslyinfusing a composition comprising ET-743 into a human having cancer at acontinuous dosage over a period up to 4 hours, wherein the step ofinfusing is repeated weekly on a cyclic basis.
 2. A method according toclaim 1, wherein the cyclic basis comprises one or more weeks of aninfusion phase, and one or more weeks of a rest phase, the rest phasenot being longer than the infusion phase.
 3. A method according to claim1, wherein the infusion time is from 1 to 3 hours.
 4. A method accordingto claim 1, wherein the infusion time is 2 to 3 hours.
 5. A methodaccording to claim 1, wherein the infusion time is about 3 hours.
 6. Amethod according to claim 1, wherein the dosage of Et-743 is below 650μg/m²/week.
 7. A method according to claim 6, wherein the dosage is from300 to 600 μg/m2/week.
 8. A method according to claim 6, wherein thedosage is from 400 to 600 μg/m²/week.
 9. A method according to claim 6,wherein the dosage is from 525 to 600 μg/m²/week.
 10. A method accordingto claim 6, wherein the dosage is about 580 μg/m²/week.
 11. A methodaccording to claim 1, wherein the cyclic basis comprises weeklyadministration and a rest phase in each cycle.
 12. A method according toclaim 11, wherein the rest period is one week within each cycle.
 13. Amethod according to claim 1, wherein each cycle is 2 to 4 weeks.
 14. Amethod according to claim 1, for the treatment of sarcoma, osteosarcoma,ovarian cancer, breast cancer, melanoma, colorectal cancer,mesothelioma, renal cancer, endometrial cancer or lung cancer.
 15. Amethod according to claim 1, wherein another drug is administered toprovide a combination therapy.
 16. A method according to claim 15,wherein the other drug is selected from: a) drugs with antimitoticeffects, especially those which target cytoskeletal elements, includingmicrotubule modulators such as taxane drugs (such as taxol, paclitaxel,taxotere, docetaxel), podophylotoxins or vinca alkaloids (vincristine,vinblastine); b) antimetabolite drugs (such as 5-fluorouracil,cytarabine, gemcitabine, purine analogues such as pentostatin,methotrexate); c) alkylating agents or nitrogen mustards (such asnitrosoureas, cyclophosphamide or ifosphamide); d) drugs which targetDNA such as the antracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; e) drugs which target topoisomerases suchas etoposide; f) hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyprotrone or octreotide;g) drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; h) alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; i) drugs potentially affecting metastasis of tumours suchas matrix metalloproteinase inhibitors; j) gene therapy and antisenseagents; k) antibody therapeutics; l) other bioactive compounds of marineorigin, notably the didemnins such as aplidine; m) steroid analogues, inparticular dexamethasone; n) anti-inflammatory drugs, includingnonsteroidal agents (such as acetaminophen or ibuprofen) or steroids andtheir derivatives in particular dexamethasone; and o) anti-emetic drugs,including 5HT-3 inhibitors (such as gramisetron or ondasetron), andsteroids and their derivatives in particular dexamethasone.
 17. A methodaccording to claim 16, wherein the other drug is selected fromdexamethasone, doxorubicin, cisplatin, paclitaxel and dexamethasone. 18.The use of ecteinascidin 743 in the preparation of a pharmaceuticalcomposition for a method according to claim
 1. 19. A medical kit foradministering ET-743, comprising printed instructions for administeringET-743 according to a method of claim 1, and a supply of ET-743 indosage units for at least one cycle, wherein each dosage unit containsan appropriate amount of ET-743 for the method and a pharmaceuticallyacceptable carrier.